1. Field of the Invention
The current invention relates to the field of real-time PCR. More particular, the current invention relates to the field of quantifying nucleic acids, in particular cellular mRNA.
2. Description of the Related Art
Quantification of mRNA has been an outstanding task in the field of molecular biology in order to obtain information on the expression of particular genes of interest. Conventionally, this has been done either by means of semi-quantitative Northern Blot analysis or semi-quantitative RNAse protection assays.
In addition, availability of the PCR technology and especially availability of Reverse Transcriptase PCR (RT-PCR), have enabled a more sensitive quantitative detection of low abundance mRNAs from small samples. In RT-PCR, a single-stranded cDNA is produced first from the mRNA to be analyzed using a reverse transcriptase. Subsequently, a double-stranded DNA amplification product is generated with the aid of PCR.
A distinction is made between two different variants of this method. In the so-called relative quantification the ratio of the expression of a certain target RNA is determined relative to the amount of RNA of a so-called housekeeping gene which is assumed to be constitutively expressed in all cells independent of the respective physiological status. Hence the mRNA is present in approximately the same amount in all cells. The advantage of this is that different initial qualities of the various sample materials and the process of RNA preparation have no influence on the particular result. However, an absolute quantification is not possible with this method.
Alternatively, the absolute amount of RNA used can be determined with the aid of standard nucleic acids of a known copy number and amplification of a corresponding dilution series of this standard nucleic acid. When using internal standards i.e. by amplifying the standard and target nucleic acid in one reaction vessel, standards have to be used that have different sequences compared to the target nucleic acid to be analyzed in order to be able to distinguish between the amplification of the standard and target nucleic acid.
Further progress could be achieved by applying methods of kinetic real-time PCR which allow for a kinetic monitoring of the amplification reaction and thus more accurate quantification of particular target molecules.
In this case, the formation of PCR products is monitored in each cycle of the PCR. The amplification is usually measured in thermocyclers which have additional devices for measuring fluorescence signals during the amplification reaction. A typical example of this is the Roche Molecular Biochemicals LightCycler (Cat. No. 20110468). The amplification products are for example detected by means of fluorescent labeled hybridization probes which only emit fluorescence signals when they are bound to the target nucleic acid or in certain cases also by means of fluorescent dyes that bind to double-stranded DNA.
A defined signal threshold is determined for all reactions to be analyzed and the number of cycles Cp required to reach this threshold value is determined for the target nucleic acid as well as for the reference nucleic acids such as the standard or housekeeping gene. The absolute or relative copy numbers of the target molecule can be determined on the basis of the Cp values obtained for the target nucleic acid and the reference nucleic acid (Gibson, U. E., et al., Genome Res 6 (1996) 995–1001.; Bieche, I., et al., Cancer Res 59 (1999) 2759–65.; WO 97/46707). Such methods are also referred to as a real-time PCR.